The goal of GenPharm's program is to develop methods for the introduction of yeast artificial chromosomes (YACs) into the germline of mice, and to apply the technology toward the generation of transgenic mice expressing a broad, fully human immunoglobulin repertoire. These mice would permit the isolation of human monoclonal antibodies in an analogous manner to rodent monoclonal antibodies. The goal of Phase I to introduce small YACs carrying V,D,J and Cmu regions of the human heavy chain immunoglobulin gene into the germline of mice to create transgenic mice carrying a human immunoglobulin minigene. The mice will be tested for expression of human IgM proteins in the serum, and for DNA rearrangement in spleen B-cells. In Phase II, the animals generated in Phase I will be bred with Human kappa light chain expressing transgenic mice, and the resultant pilot mouse tested for its capacity to express a primary repertoire of surface and secreted fully human IgMs. Also, additional constant and variable regions will be cloned in YACs to add to or replace the Phase I YACs, to permit class switching, somatic mutation and IgG production. If necessary, sequence modifications such as the introduction of additional enhancer elements will also be added. Transgenic mice will also be generated carrying large fragments of the unrearranged human kappa gene and bred to the Phase II heavy chain mice. Finally, these mice will be bred with mice lacking functional endogenous immunoglobulin genes. Hybridomas will be generated at various stages of the project from their offspring to determine if somatic mutation of the transgene can efficiently occur, leading to a broad repertoire of high affinity, fully human IgG producing B cells.